Figure 4

Replicate experimental infections show variation for proportions of parasites showing caspase-like activity and viability. Two sets of replicate experiments were set up by infecting 10 male MF1 mice 8-10 weeks old per replicate with 10⁷ P. berghei parasites after pre-treatment with Phenylhydrazine 2 days pre-infection (120 mg/kg). One mouse from experiment 1 failed to become infected so was removed from the study. Cultures were then set up 4 days post infection. After 18 hours ookinetes were purified using MACS LS cell separation columns (Miltenyi biotec) and a minimum of 30 parasites were assayed for caspase like activity and viability using CaspaTag™ Pan-Caspase In Situ Assay Kit, Fluorescein in conjunction with propidium iodide (Chemicon international, USA). Green indicates healthy ookinetes negative for caspase-like activity with intact membranes, yellow indicates early apoptotic ookinetes displaying caspase-like activity with intact membranes, orange indicates late apoptotic ookinetes displaying caspase-like activity but also compromised membranes and red indicates dead cells with compromised membranes [25]. More detailed methodology is available in Additional file 1.